Worldwide diabetes is projected to reach epidemic proportions in the next 25 years, however, the prevalence of diabetes in the U.S., India, China, Russia, and Japan is already spiraling out of control. One promising therapy for diabetic conditions linked to the functional or outright loss of insulin-producing cells (i.e. pancreatic beta cells) is simply replenishing them. Two main strategies are being developed: 1) Transplantation of beta cells (cell replacement), and 2) Stimulation of endogenous progenitor/adult stem cells (cellular regeneration). Combined with suppression of an autoimmune response which targets beta cells, such therapies could represent lasting cures. The ultimate goal of this project is to create a diabetes model in which genetic networks that regulate the regeneration of insulin-producing pancreatic beta cells in vertebrates can be elucidated. Because zebrafish have a remarkable capacity for cellular regeneration - and are amenable to forward genetics - mutations can be identified which disrupt the regenerative process. Accordingly, we have adapted an inducible cellular ablation system, termed ZAP, toward the goal of creating new tools for the study of cellular regeneration in zebrafish. The ZAP system can be targeted to any genetically definable cellular subtype and thereby model diseases linked to the loss of a particular cell type (e.g., Type I diabetes). The ZAP system is based on transgenic expression of a fusion protein between a pro-drug converting enzyme and a fluorescent reporter. The enzyme converts otherwise innocuous pro-drugs into cytotoxins, thus adding pro-drugs to the water induces ablation in multiple fish simultaneously. The reporter allows automated quantitative detection of the presence or absence of the targeted cell type. We have recently demonstrated two key findings using transgenic zebrafish expressing ZAPs specifically in beta cells: 1) ZAP-expressing beta cells can be specifically eliminated upon treatment with pro-drug, 2) Upon removal of pro-drug, beta cells are rapidly regenerated over the course of the next few days. Here we propose to create transgenic zebrafish expressing the ZAP system in beta cells. In Phase II efforts, these transgenic animals will be mutagenized and screened for those individuals in which this innate capacity for beta cell regeneration has been disrupted. These mutant lines will provide unique insights into the genetic circuitry underlying the regulation of beta cell regeneration and may be utilized in downstream commercialization efforts for the efficient screening and identification of compounds which stimulate progenitor/stem cells to replace lost beta cells. The specific aims of this proposal are: 1) The creation of transgenic lines that will facilitate ratiometric automated detection of targeted (beta) and control cells (alpha), and 2) Optimization and scaling of quantitative automated detection methods. PUBLIC HEALTH RELEVANCE: The number of people diagnosed with diabetes will reach epidemic proportions worldwide within the first quarter of this century. Therapies aimed at replacing or amplifying the number of insulin-producing cells in the body (the "beta" cells of the pancreas) show great promise for diabetic conditions which require daily insulin injections. This study proposes to create a series of diabetic disease model organisms. Future studies will utilize these models to identify the genetic networks that regulate the process of beta cell regeneration. Insights gained from generating a diabetes model will help indentify drugs capable of safely stimulating the production of new insulin-producing beta cells - a potential cure for this debilitating disease.